Natural xylanase enzymes, such as that of the fungus Trichoderma reesei, have been added to animal feed to increase the efficiency of digestion and assimilation of nutrients. During digestion of feed grains such as wheat and barley, non-starch polysaccharides, including xylan, increases the viscosity of the digesta in the absence of added exogenous enzyme. This interferes with the diffusion of the digestive enzymes to the feed and the subsequent assimilation of the nutrients. The highly viscous digesta increases the occurrence of sticky stool, which increases the likelihood of disease and causes effluent run-off problems. The addition of xylanase in feed breaks down the xylan and decreases the viscosity of the digesta, thereby helping to alleviate these problems. Xylanase produces a cost saving by increasing the efficiency of feed conversion. Xylanase can decrease the feed consumed/weight gain ratio by 5–15% (Viveros, A., Brenes, A., Pizarro, M. and Castano, M., 1994, Animal Feed Sci. Technol. 48:237–251).
Xylanase enzymes used for feed are typically aqueous solutions of active protein, stabilizers, preservatives and other additives. The enzymes are typically sprayed onto the feed at concentration of 100–2000 ml per tonne feed. Alternatively, granular or powdered xylanase can be used. Once the feed is consumed by the animal, the enzyme acts on xylan as the feed is ingested and digested in the gut. Eventually the xylanase, a protein molecule, is hydrolysed by the digestive enzymes (proteases) into amino acids like any protein in the feed.
Increasingly, animal feeds are pelleted at high temperatures for sterilization against harmful bacteria, for example Salmonella. Feed pelleting is carried out by heating the feed solids with 100 to 140° C. steam and passing them through an extruder/pelleting auger to form feed pellets, which then cool in a storage bin. The typical time required for the material to pass through the system is 30 minutes. As is known in the art, higher temperatures can be used with shorter pelleting times, and lower temperatures with longer pelleting times, provided that the necessary moisture levels are obtained. The overall resulting temperature within the solids, prior to, during, and after pellet formation reaches about 70–95° C., for up to 30 min. It is desirable to add the xylanase during the feed pelleting process. This would save the feed formulators the additional step of adding liquid xylanase, which is inconvenient and can introduce microbial contamination into the feed. The option of adding solid xylanase as a separate step is also undesirable, as the solids would not be evenly mixed. Marquardt and Bedford (1997, Enzymes in Poultry and Swine Nutrition, Marquardt R. R. and Han Z. eds., pp. 129–138) indicate that even though currently available enzymes are beneficial for use as feed additives, new enzymes exhibiting high activity and resistance to heat treatment are also desired, however, they note that enzymes exhibiting these properties are not available.
Xylanases of Family 11 (also termed Family G xylanases) have several properties suitable for feed applications due to their small size and high activity. An example of a moderate temperature Family 11 xylanases is TrX, which is obtained from Trichoderma reesei. Moderate temperature xylanases are proven feed additive enzymes with temperature and pH optima compatible with the physiological conditions in the digestive system of animals. However, these enzymes can not tolerate the high temperature of the pelleting process and become inactive during this step.
Xylanases from high temperature microorganisms (eg. a thermophile), for example Thermomonospora fusca xylanase (termed TfX, also a Family 11 xylanase), have also been considered for feed pelleting. The thermostability of such enzymes is sufficient to tolerate the pelleting temperatures. However, thermophilic xylanases have optimum activity at high temperatures (70–80° C.), and several of these enzymes have a high pH optimum of 7–9. When introduced into the digesting system of an animal, with a physiological temperature of around 40° C. (e.g. poultry 43° C., a similar temperature is noted within swine) and pH of 3–5 in the digesta, these enzymes function poorly.
Family 11 xylanases have been modified by protein engineering to improve the properties of these enzymes for industrial applications. These modifications have been directed at increasing the temperature and pH optima, along with the thermostability, of these enzymes for specific applications. For example, U.S. Pat. No. 5,405,769 (WO 94/24270) is directed to site-specific mutagenesis of Bacillus circulans xylanase (BcX) for the improvement of the thermostability of this enzyme. The disclosed modifications relate to the formation of intermolecular and intramolecular disulfide bonds within BcX, and these modifications resulted in increased thermostability. For example, an improvement in thermostability of up to 6° C. with the addition of a single disulfide bond, and up to 10° C. with two disulphide bonds was observed. Other modifications included linking the N- and C-termini which increased thermostability by 6° C., or N-terminal mutations, which increased thermostability by 2° C. However, with all of the above modifications the resultant enzymes were either less active (up to 45% less active), or exhibited an increase in the temperature and pH optima. As such these enzymes are not suitable for feed pelleting applications.
U.S. Pat. No. 5,759,840 also discloses modifications to BcX and Trichoderma reesei xylanase (TrX) to increase the thermostability, while at the same time increase the temperature and pH optima of these enzymes. Again, these xylanases would not be suitable for feed pelleting applications.
The above results are in agreement with other reports that note that disulfide bonds are not among the thermostabilization mechanisms employed by thermophilic enzymes (Cowan, D. A., 1995, Essays Biochem. 29:193–207), as the disulfide can be broken into dehydroalanine and thiocysteine at temperatures over 80° C. Therefore, the enhancement of stability of an enzyme using disulfide bonds is limited to lower temperature ranges. The disulfide bond is thus not recommended to improve the stability of the enzyme at high temperatures (Gupta, M. N., 1991, Biotech. Applied Biochem. 14:1–11; Cowan, D. A., 1995, Essays Biochem. 29:193–207).
None of the above documents address methods for obtaining xylanase enzymes using conventional screening techniques, or by modifying xylanase enzymes, that exhibit the properties of higher temperature tolerance while maintaining optimal performance under conditions of physiological pH and temperature.
An improvement in the thermostability of Trichoderma reesei xylanase II was reported by Paloheimo et al (Paloheimo, M., Mantyla, A., Vehmaanpera, J., Hakola, S., Lantto, R., Lahtinen, T., Parkkinen, E., Fagerstrom, R. and Suominen, P. 1997, in Carbohydrases from Trichoderma reesei and Other Microorganisms p255–264). Of the five mutants characterized, the most improved mutant (glutamic acid-38 TrX) retained 50% of activity at 57° C. after 9 min, as compared to 7 min by wide type TrX. Arase et al (Arase, A., Yomo, T., Urabe, I., Hata, Y., Katsube, Y. and Okada, H., 1993, FEBS Lett. 316:123–127) describes several modifications to improve the thermostability of a Bacillus pumilis xylanase (BpX), however only up to 40% of the residual enzymatic activity was maintained following incubation of these enzymes at a temperature of 57° C. for 20 min. Even though, in both of these studies the effects of increased thermostability on pH and temperature optima of the enzymes were not determined, these enzymes exhibit inadequate thermostability for feed pelleting applications.
In spite of a wide range of experience in screening, testing and modifying xylanase enzymes, there are no reports of xylanases that exhibit the combination of properties required for feed pelleting applications: high thermostability, with optimum activity at physiological pH and temperature. No natural xylanases have been selected, nor has any mutation methodology for the Family 11 xylanases been developed to increase thermostability of xylanase enzymes to, without any change in the temperature and pH optima, and a concomitant loss of the specific activity of the enzyme. Such selected natural xylanases, or xylanases prepared using mutation methodology would offer the advantages of enhancement of feed digestibility and processing in pelleting.
The present invention is directed to obtaining xylanase enzymes that exhibit the property of increased thermostability, while maintaining pH and temperature optima that are typically found under physiological conditions.
It is an object of the invention to overcome disadvantages of the prior art.
The above object is met by the combinations of features of the main claims, the sub-claims disclose further advantageous embodiments of the invention.